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Because the artificial cultivation environment differs from the natural habitat of the Bohai Sea, it is essential to study the adaptability of hatchery-reared juveniles released into this area. Hatchery-origin Paralichthys olivaceus stocks were identified via morphological observation and genetic tags (i.e., mitochondrial DNA and microsatellites). Seven genomic microsatellite loci with a high PIC (polymorphic information content, 0.885–0.945) and a high exclusion probability (0.930–0.981) were selected and arranged into two multiplex PCRs. In total, 46.75% of the captures were assigned to our broodstocks. There were no significant differences in genetic diversity, such as Ar (allelic richness, P = .754) and He (expected heterozygosity, P = 0. 658), among the hatchery-origin stock (HS), their parents (P), the wild stock (WS) and the captured broodstocks (CB) in 2016. However, losses of Ar in the HS indicated that more attention should be given to the number of broodstocks in a reared population. The overall genetic divergence among these four groups was low (FST = 0.005), and the results of AMOVA also showed no genetic differences among them (P = .500), indicating that no pronounced genetic differentiation was found following the stock enhancement program in the Bohai Sea. There was no significant difference between HS and WS in terms of growth parameters (P > .05) and feeding ecology (P > .05). Furthermore, the stomach contents of the two groups were basically identical and consisted mainly of fish. Therefore, overall, the hatchery-origin juveniles had already adapted to the natural area after the summer fishing moratorium.

Germ cell transplantation (GCT) is a promising reproductive technology in animals, including teleost fish. Here, we established a GCT procedure in Japanese flounder (Paralichthys olivaceus). Two-year-old male Japanese flounder were treated with temperature (28 °C) and busulfan (two doses, 18 mg/kg) to deplete endogenous germ cells and were then used as recipients. The discontinuous Percoll gradient purified oogonia were used as donor germ cells and transplanted into the recipients' testes through the genital pore. Histological and fluorescent observations showed the colonization, proliferation, and differentiation of transplanted oogonia in the testes of recipient fish. Ten months after GCT, 16 of 17 recipients produced sperm, and among them, two recipients produced donor-derived sperm, which was confirmed by microsatellite genotyping. The sperm of these two recipients were used to fertilize the eggs of wild-type females, and the donor-derived germline transmission rates were 45.5% and 43.0%, respectively, based on the microsatellite paternity analysis of the hatched larvae. Our established protocol can be applied to accelerate the reproduction cycle and be used for generating valuable surrogate breeding material and conserving the germplasm resources of Japanese flounder.

Clonal fishes are useful tools in biology and aquaculture studies due to their isogenicity. In Japanese flounder (Paralichthys olivaceus), a group of homozygous clones was created by inducing meiogynogenesis in eggs from a mitogynogenetic homozygous diploid. As the clones reached sexual maturity, meiogynogenesis was again induced in order to produce a 2nd generation clonal group of Japanese flounder. After 3 months, there were 611 healthy, surviving individuals. Twenty-four microsatellite markers, that covered all the linkage groups of Japanese flounder, were used to identify the homozygosity of the 2nd generation clones; no heterozygous locus was detected. This indicates that the production of a 2nd generation clonal group of Japanese flounder was successful. Restriction-site DNA associated sequencing at the genomic level also confirmed the homozygosity and clonality of the 2nd generation clonal group. Furthermore, these 2nd generation clones had a small coefficient of variation for body shape indices at 210 days of age and showed a high degree of similarity in body characteristics among individuals. The successful production of 2nd generation clones has laid the foundation for the large-scale production of clonal Japanese flounder.

Sterility is a serious problem that can affect all bionts. In teleosts, double haploids (DHs) induced by mitogynogenesis are often sterile. This sterility severely restricts the further application of DHs for production of clones, genetic analysis, and breeding. However, sterile DH individuals are good source materials for investigation of the molecular mechanisms of gonad development, especially for studies into the role of genes that are indispensable for fish reproduction. Here, we used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation, we obtained 52,474 contigs and found that 60.7% of these shared homologies with existing sequences. A total of 1225 differentially expressed unigenes were found, including 492 upregulated and 733 downregulated genes. Gene Ontology and KEGG analyses showed that genes showing significant upregulation, such as CYP11A1, CYP11B2, CYP17, CYP21, HSD3β, bcl2l1, and PRLR, principally correlated with sterol metabolic process, steroid biosynthetic process, and the Jak-stat signaling pathway. The significantly downregulated genes were primarily associated with immune response, antigen processing and presentation, cytokine-cytokine receptor interaction, and protein digestion and absorption. Using a co-expression network analysis, we conducted a comprehensive comparison of gene expression in the gonads of fertile and sterile female DH Japanese flounder. Identification of genes showing significantly different expression will provide further insights into DH reproductive dysfunction and oocyte maturation processes in teleosts.

Sterility is a serious problem that can affect all bionts. In teleosts, double haploids (DHs) induced by mitogynogenesis are often sterile. This sterility severely restricts the further application of DHs for production of clones, genetic analysis, and breeding. However, sterile DH individuals are good source materials for investigation of the molecular mechanisms of gonad development, especially for studies into the role of genes that are indispensable for fish reproduction. Here, we used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation, we obtained 52,474 contigs and found that 60.7% of these shared homologies with existing sequences. A total of 1225 differentially expressed unigenes were found, including 492 upregulated and 733 downregulated genes. Gene Ontology and KEGG analyses showed that genes showing significant upregulation, such as CYP11A1, CYP11B2, CYP17, CYP21, HSD3β, bcl2l1, and PRLR, principally correlated with sterol metabolic process, steroid biosynthetic process, and the Jak-stat signaling pathway. The significantly downregulated genes were primarily associated with immune response, antigen processing and presentation, cytokine-cytokine receptor interaction, and protein digestion and absorption. Using a co-expression network analysis, we conducted a comprehensive comparison of gene expression in the gonads of fertile and sterile female DH Japanese flounder. Identification of genes showing significantly different expression will provide further insights into DH reproductive dysfunction and oocyte maturation processes in teleosts.